Regeneration of the antioxidant ubiquinol by flavoenzymes and the role of antioxidant defence in experimental hepatocarcinogenesis

Abstract: In the environment, to which we have adapted and thrive, we are constantly exposed to reactive molecules with the potential to cause intracellular damage and cellular malfunction. One of the molecules that is transformed to very reactive free radicals is oxygen that can give rise to a variety of reactive oxygen species formed in close vicinity to sensitive structures in different compartments of the cell. This is a potential threat to cell function and cell survival at the same time as it gives possibilities for change and evolution in a positive sense over time when it happens at the right time in the right cell. To protect the cell from the constant attacks of free radicals a series of cell defence mechanisms has been developed through evolution. Among them are a number of antioxidants that are either synthesised in the cell or taken up from the food. To persevere the stream of reactive molecules the antioxidants have to be regenerated by renewable redox-reactions. This thesis concerns studies of ubiquinol, the only endogenously produced lipid soluble antioxidant known so far and the enzymes and reactions involved in the regeneration of ubiquinol from its oxidized form, ubiquinone, in extramitochondrial compartments of the cell. We showed that the flavoenzyme lipoamide dehydrogenase could reduce ubiquinone to its reduced form ubiquinol and that the rate of this reaction was elevated 10-fold when zinc was added to the reaction mixture and when NADPH was used as cofactor. The reduction of ubiquinone by lipoamide dehydrogenase was further characterised and an interesting observation was that the pH-optimum of this reaction was 6 and 5.5 in the presence and absence of zinc, respectively. The ability to reduce ubiquinone was also studied with another flavoenzyme, thioredoxin reductase, and it was shown that under normal physiological conditions this enzyme has the highest activity of all extramitochondrial enzymes so far studied. We also demonstrated that this reaction was selenium dependent. The regeneration of ubiquinol by cytosolic proteins isolated from both normal rat liver and neoplastic tissue. The reaction rate in the cytosol isolated from neoplastic tissue was increased by 50% compared to control when NADPH was used as cofactor. It was also demonstrated that 90% of the activity was in the protein fraction with a molecular weight of above 100 kDa. The properties of the reaction was similar with those of thioredoxin reductase suggesting that this enzyme is the one mainly responsible for the regeneration of ubiquinol in the cytosol. In paper V it was for the first time described that ubiquinone was induced by carbon tetrachloride treatement. In conclusion these findings suggest that the antioxidant ubiquinol, in extramitochondrial compartments, is mainly regenerated by the flavoenzymes thioredoxin reductase and lipoamide dehydrogenase. The reactions responsible for regeneration of antioxidants are of great importance for cell survival and adaptation to environmental stress situations.